Rapid Complexation of Aptamers by Their Specific Antidotes.

Nucleic acid ligands, aptamers, harbor the unique characteristics of small molecules and antibodies. The specificity and high affinity of aptamers enable their binding to different targets, such as small molecules, proteins, or cells. Chemical modifications of aptamers allow increased bioavailability. A further great benefit of aptamers is the antidote (AD)-mediated controllability of their effect. In this study, the AD-mediated complexation and neutralization of the thrombin binding aptamer NU172 and Toll-like receptor 9 (TLR9) binding R10-60 aptamer were determined. Thereby, the required time for the generation of aptamer/AD-complexes was analyzed at 37 °C in human serum using gel electrophoresis. Afterwards, the blocking of aptamers' effects was analyzed by determining the activated clotting time (ACT) in the case of the NU172 aptamer, or the expression of immune activation related genes IFN-1ß, IL-6, CXCL-10, and IL-1ß in the case of the R10-60 aptamer. Gel electrophoresis analyses demonstrated the rapid complexation of the NU172 and R10-60 aptamers by complementary AD binding after just 2 min of incubation in human serum. A rapid neutralization of anticoagulant activity of NU172 was also demonstrated in fresh human whole blood 5 min after addition of AD. Furthermore, the TLR9-mediated activation of PMDC05 cells was interrupted after the addition of the R10-60 AD. Using these two different aptamers, the rapid antagonizability of the aptamers was demonstrated in different environments; whole blood containing numerous proteins, cells, and different small molecules, serum, or cell culture media. Thus, nucleic acid ADs are promising molecules, which offer several possibilities for different in vivo applications, such as antagonizing aptamer-based drugs, immobilization, or delivery of oligonucleotides to defined locations.

Saving or Subordinating Life? Popular Views in Israel and Germany of Donor Siblings Created through PGD.

To explore how cultural beliefs are reflected in different popular views of pre-implantation genetic diagnosis for human leukocyte antigen match (popularly known as "savior siblings"), we compare the reception and interpretations, in Germany and Israel, of the novel/film My Sister's Keeper. Qualitative analysis of reviews, commentaries and posts is used to classify and compare normative assessments of PGD for HLA and how they reproduce, negotiate or oppose the national policy and its underlying cultural and ethical premises. Four major themes emanated from the comparison: loss of self-determination and autonomy; loss of dignity through instrumentalization; eugenics and euthanasia; and saving life. In both countries, most commentaries represented a dominant position, with a few negotiated positions. We also highlight the decoding of a relatively less explored bioethical aspect of My Sister's Keeper's narrative, namely the meaning of euthanasia. We conclude by discussing how the findings relate to attempts of providing cultural explanations for the regulation of HLA-PGD.

Use of synthetic single-stranded oligonucleotides as artificial test soiling for validation of surgical instrument cleaning processes.

Surgical instruments are often strongly contaminated with patients' blood and tissues, possibly containing pathogens. The reuse of contaminated instruments without adequate cleaning and sterilization can cause postoperative inflammation and the transmission of infectious diseases from one patient to another. Thus, based on the stringent sterility requirements, the development of highly efficient, validated cleaning processes is necessary. Here, we use for the first time synthetic single-stranded DNA (ssDNA_ODN), which does not appear in nature, as a test soiling to evaluate the cleaning efficiency of routine washing processes. Stainless steel test objects were coated with a certain amount of ssDNA_ODN. After cleaning, the amount of residual ssDNA_ODN on the test objects was determined using quantitative real-time PCR. The established method is highly specific and sensitive, with a detection limit of 20 fg, and enables the determination of the cleaning efficiency of medical cleaning processes under different conditions to obtain optimal settings for the effective cleaning and sterilization of instruments. The use of this highly sensitive method for the validation of cleaning processes can prevent, to a significant extent, the insufficient cleaning of surgical instruments and thus the transmission of pathogens to patients.

In vivo tissue engineering: mimicry of homing factors for self-endothelialization of blood-contacting materials.

Thrombogenicity of foreign surfaces is the major obstacle in cardiovascular interventions. Despite enormous advances in biomaterials research, the hemocompatibility of blood-contacting materials is still not satisfactory and the native endothelium still represents the ideal surface for blood contact. Circulating adult endothelial progenitor cells (EPCs) in the human blood provide an excellent source of autologous stem cells for the in vivo self-endothelialization of blood-contacting materials. For this purpose, material surfaces can be coated with capture molecules mimicking natural homing factors to attract circulating EPCs. Hitherto, several ligands, such as aptamers, monoclonal antibodies, peptides, selectins and their ligands, or magnetic molecules, are used to biofunctionalize surfaces for the capturing of EPCs directly from patient's bloodstream onto blood-contacting materials. Subsequently, attracted EPCs can differentiate into endothelial cells and generate an autologous endothelium. The in vivo self-endothelialization of blood-contacting materials prevents the recognition of them as a foreign body; this opens up revolutionary new prospects for future clinical stem-cell and tissue engineering strategies.

Application of a rotating bioreactor consisting of low-cost and ready-to-use medical disposables for in vitro evaluation of the endothelialization efficiency of small-caliber vascular prostheses.

The incomplete endothelialization of especially small-caliber vascular prostheses after implantation in patients is a major disadvantage in cardiovascular interventions. The lack of an endothelium leads to the occurrence of thrombosis at the luminal surface of artificial vascular prostheses. Thus, the development of new graft materials and coatings for induction of complete endothelialization on the implant surfaces is a promising approach to improve hemocompatibility and maintain long-term graft patency. In this study, we designed a rotation model to evaluate the early endothelial cell (EC) seeding efficiency of different small-caliber vascular devices, such as stents and vascular grafts. The suitability of the designed rotation model for endothelialization studies was investigated by seeding and cultivation of prostheses with ECs followed by scanning electron microscopy. Furthermore, the viability of attached ECs was determined by calcein acetoxymethyl ester (AM) staining. The rotation model consisting of low-cost medical disposables enabled sterile incubation and cultivation of ECs with vascular devices. Simultaneously, the rotation of the bioreactor ensured a uniform distribution and adhesion of cells to the devices. Calcein AM staining of adherent cells on prostheses revealed excellent cell viability. Moreover, using the designed rotation model, an influence of different coatings and materials on the adhesion and spreading of ECs was demonstrated. The rotating bioreactor described and used in this study not only saves time and money but is also eminently useful for the accelerated preclinical evaluation of the endothelialization efficiency of different materials and surface coatings of small-caliber vascular devices.

Absolute quantification of cell-bound DNA aptamers during SELEX.

In the fields of diagnosis, imaging, regenerative medicine, and drug targeting, aptamers are promising nucleic acid ligands for specific recognition and binding of whole living cells. These aptamers are selected by a combinatorial chemistry technique called cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment). During this iterative procedure of in vitro selection and enzymatic amplification, the enrichment of cell binding aptamers is generally monitored by flow cytometry. This method needs the use of fluorophore-labeled oligonucleotides for detection and allows only the relative evaluation of the aptamer binding compared with the control. Here, we describe the development and validation of a new quantitative real time polymerase chain reaction (qPCR) method for the absolute determination of cell bound aptamers during cell-SELEX. The method is based on SYBR Green I real-time PCR technology and uses an aptamer standard curve to determine the accurate aptamer amount on cells after the incubations. Lysates of cells with bound aptamers were used to identify the absolute amount of aptamers on cells. This method is highly sensitive and allows the detection of very small quantities of aptamers in cell lysate samples. The lower detection limit is 20 fg. The established qPCR method can be used as an additional monitoring tool during cell-SELEX to determine the enrichment of cell binding aptamers on cells, whereby the absolute quantity is determined. Furthermore, the contamination of the amplified aptamer pool with by-products can be prevented by prior determination of bound aptamer amount on cells.